Biology subjects

David, L. L.

Publications and source records attributed to David, L. L..

6 recordsLinked to original sources

bioRxiv2024

Analysis of mouse lens morphological and proteomic abnormalities following depletion of βB3-crystallin

Crystallin proteins serve as both essential structural and as well as protective components of the ocular lens and are required for the transparency and light refraction properties of the organ. The mouse lens crystallin proteome is represented by A-, B-, {beta}A1-, {beta}A2-, {beta}A3-, {beta}A4-, {beta}B1-, {beta}B2-, {beta}B3-, {gamma}A-, {gamma}B-, {gamma}C-, {gamma}D-, {gamma}E, {gamma}F-, {gamma}N-, and {gamma}S-crystallin proteins encoded by 16 genes. Their mutations are responsible for lens opacification and early onset cataract formation. While many cataract-causing missense and nonsense mutations are known for these proteins, including the human CRYBB3 gene, the mammalian loss-of function model of the Crybb3 gene remains to be established. Herein, we generated the first mouse model via deletion of the Crybb3 promoter that abolished expression of the {beta}B3-crystallin. Histological analysis of lens morphology using newborn {beta}B3-crystallin-deficient lenses revealed disrupted lens morphology with early-onset phenotypic variability. In-depth lens proteomics at four time points (newborn, 3-weeks, 6-weeks, and 3-months) showed both down- and up-regulation of various proteins, with the highest divergence from control mice observed in 3-months lenses. Apart from the {beta}B3-crystallin, another protein Smarcc1/Baf155 was down-regulated in all four samples. In addition, downregulation of Hspe1, Pdlim1, Ast/Got, Lsm7, Ddx23, and Acad11 was found in three time points. Finally, we show that the {beta}B3-crystallin promoter region, which contains multiple binding sites for the transcription factors AP-2, c-Jun, c-Maf, Etv5, and Pax6 is activated by FGF2 in primary lens cell culture experiments. Together, these studies establish the mouse Crybb3 loss-of-function model and its disrupted crystallin and non-crystallin proteomes.

developmental biology↗

bioRxiv2022

PRMT5 as a Novel Druggable Vulnerability for EWSR1-ATF1-driven Clear Cell Sarcoma

Clear cell sarcoma of soft tissue (CCSST) is an ultra-rare sarcoma with poor prognosis presently with no cure. It is characterized by a balanced t(12;22) (q13;q12) chromosomal translocation, resulting in a fusion of the Ewings sarcoma gene EWSR1 with activating transcription factor 1 (ATF1) to give an oncogene EWSR1-ATF1. Unlike normal ATF1, whose transcription activity is dependent on phosphorylation, EWSR1-ATF1 is constitutively active to drive ATF1-dependent gene transcription to cause tumorigenesis. No EWSR1-ATF1-targeted therapies have been identified due to the challenges in targeting intracellular transcription factors. To identify potential druggable targets for CCSST, we show that protein arginine methyltransferase 5 (PRMT5) is a novel enzyme in enhancing EWSR1-ATF1-mediated gene transcription to sustain CCSST cell proliferation. Genetic silencing of PRMT5 in CCSST cells resulted in severely impaired cell proliferation and EWSR1-ATF1-driven transcription. Furthermore, the clinical-stage PRMT5 inhibitor JNJ-64619178 potently and efficaciously inhibited CCSST cell growth in vitro and in vivo. These results provide new insights into PRMT5 as a transcription regulator and warrant JNJ-64619178 for further clinical development to treat CCSST patients.

cancer biology↗

bioRxiv2020

Accumulative deamidation of human lens protein γS-crystallin leads to partially unfolded intermediates with enhanced aggregation propensity

Age-related cataract is a major cause of blindness worldwide. Yet, the molecular mechanisms whereby large, light scattering aggregates form is poorly understood, because of the complexity of the aggregates isolated from human lenses. The predominant proteins in the lens are structural proteins called crystallins. The {gamma}S-crystallin is heavily modified in cataractous lenses by deamidation, which introduces a negative charge at labile asparagine residues. The effects of deamidation at asparagines, N14, N76, and N143, were mimicked by replacing the asparagine with aspartate using site-directed mutagenesis. The effects of these surface deamidations on the stability, unfolding, and aggregation properties of {gamma}S were determined using dynamic light scattering, chemical and thermal-denaturation, and hydrogen-deuterium exchange with mass spectrometry. We found that a small population of all the deamidation mimics aggregated directly into large light scattering bodies with a radius greater than 10 nm that contributed 14-60% of the total scattering intensity compared to 7% for WT under the same conditions. A possible mechanism was identified under partially denaturing conditions, where deamidation led to significantly more rapid unfolding and aggregation particularly for N76D compared to WT. The triple mutant was further destabilized, reflecting the enhanced aggregation properties of N14D and N143D. Thus, the effects of deamidation were both site-specific and cumulative. A-crystallin was ineffective at acting as a chaperone to prevent the aggregation of destabilized, deamidated {gamma}S. It is concluded that surface deamidations, while causing minimal structural disruption individually, progressively destabilize crystallin proteins, leading to their unfolding and precipitation in aged and cataractous lenses.

biochemistry↗

bioRxiv2019

Cataract-associated deamidations on the surface of γS-crystallin increase protein unfolding and flexibility at distant regions

Deamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from cataractous lenses. However, the mechanism by which deamidation causes proteins to become insoluble is not known, because of only subtle structural changes observed in vitro. We have identified Asn14 and Asn76 of {gamma}S-crystallin as highly deamidated in insoluble proteins. These sites are on the surface of the N-terminal domain and were mimicked by replacing the Asn with Asp residues. We used heteronuclear NMR spectroscopy to measure their amide hydrogen exchange and 15N relaxation dynamics to identify regions with significantly increased dynamics compared to wildtype-{gamma}S. Changes in dynamics were localized to the C-terminal domain, particularly to helix and surface loops distant from the mutation sites. Thus, a potential mechanism for {gamma}S deamidation-induced insolubilization in cataractous lenses is altered dynamics due to local regions of unfolding and increased flexibility.

biophysics↗

bioRxiv2019

Identification of PKC -dependent phosphoproteins in mouse retina

Adjusting to a wide range of light intensities is an essential feature of retinal rod bipolar cell (RBC) function. While persuasive evidence suggests this modulation involves phosphorylation by protein kinase C-alpha (PKC), the targets of PKC phosphorylation in the retina have not been identified. PKC activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific PKC antibody and antibodies to phosphorylated PKC motifs. PKC activity was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. PKC-dependent retinal phosphoproteins were identified using a phosphoproteomics approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and PKC knockout (PKC-KO) mouse retinas. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to PKC-KO samples. The differentially phosphorylated proteins fall into the following functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein). Two strongly differentially expressed phosphoproteins, BORG4 and TPBG, were localized to the synaptic layers of the retina, and may play a role in PKC-dependent modulation of RBC physiology. Data are available via ProteomeXchange with identifier PXD012906.\n\nSignificanceRetinal rod bipolar cells (RBCs), the second-order neurons of the mammalian rod visual pathway, are able to modulate their sensitivity to remain functional across a wide range of light intensities, from starlight to daylight. Evidence suggests that this modulation requires the serine/threonine kinase, PKC, though the specific mechanism by which PKC modulates RBC physiology is unknown. This study examined PKC phosophorylation patterns in mouse rod bipolar cells and then used a phosphoproteomics approach to identify PKC-dependent phosphoproteins in the mouse retina. A small number of retinal proteins showed significant PKC-dependent phosphorylation, including BORG4 and TPBG, suggesting a potential contribution to PKC-dependent modulation of RBC physiology.\n\nHighlightsO_LIPKC is a major source of phosphorylation in retinal RBC dendrites and its activity in RBCs is light dependent.\nC_LIO_LIProteins showing differential phosphorylation between phorbol ester-treated wild type and PKC-KO retinas belong to the following major functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein).\nC_LIO_LIThe PKC-dependent phosphoproteins, BORG4 and TPBG, are present in the synaptic layers of the retina and may be involved in PKC-dependent modulation of RBC physiology.\nC_LI

neuroscience↗

bioRxiv2017

ELMOD1 stimulates ARF6-GTP hydrolysis to stabilize apical structures in developing vestibular hair cells

Sensory hair cells require control of physical properties of their apical plasma membranes for normal development and function. Members of the ARF small GTPase family regulate membrane trafficking and cytoskeletal assembly in many cells. We identified ELMOD1, a guanine nucleoside triphosphatase activating protein (GAP) for ARF6, as the most highly enriched ARF regulator in hair cells. To characterize ELMOD1 control of trafficking, we used a mouse strain lacking functional ELMOD1 (roundabout; rda). In rda/rda mice, cuticular plates of utricle hair cells initially formed normally, then degenerated after postnatal day 5 (P5); large numbers of vesicles invaded the compromised cuticular plate. Hair bundles initially developed normally, but the cells apical membrane lifted away from the cuticular plate, and stereocilia elongated and fused. Membrane trafficking in type I hair cells, measured by FM1-43 dye labeling, was altered in rda/rda mice. Consistent with the proposed GAP role for ELMOD1, the ARF6 GTP/GDP ratio was significantly elevated in rda/rda utricles as compared to controls, and the level of ARF6-GTP was correlated with the severity of the rda/rda phenotype. These results suggest that conversion of ARF6 to its GDP-bound form is necessary for final stabilization of the hair bundle.\n\nSignificance StatementAssembly of the mechanically sensitive hair bundle of sensory hair cells requires growth and reorganization of apical actin and membrane structures. Hair bundles and apical membranes in mice with mutations in the Elmod1 gene degenerate after formation, suggesting that the ELMOD1 protein stabilizes these structures. We show that ELMOD1 is a GTPase-activating protein in hair cells for the small GTP-binding protein ARF6, known to participate in actin assembly and membrane trafficking. We propose that conversion of ARF6 into the GDP-bound form in the apical domain of hair cells is essential for stabilizing apical actin structures like the hair bundle and ensuring that the apical membrane forms appropriately around the stereocilia.

neuroscience↗